The precise mechanism by which oral infection contributes to the pathogenesis of extra-oral diseases remains unclear. Here, we report that periodontal inflammation exacerbates gut inflammation in vivo. Periodontitis leads to expansion of oral pathobionts, including Klebsiella and Enterobacter species, in the oral cavity. Amassed oral pathobionts are ingested and translocate to the gut, where they activate the inflammasome in colonic mononuclear phagocytes, triggering inflammation. In parallel, periodontitis results in generation of oral pathobiont-reactive Th17 cells in the oral cavity. Oral pathobiont-reactive Th17 cells are imprinted with gut tropism and migrate to the inflamed gut. When in the gut, Th17 cells of oral origin can be activated by translocated oral pathobionts and cause development of colitis, but they are not activated by gut-resident microbes. Thus, oral inflammation, such as periodontitis, exacerbates gut inflammation by supplying the gut with both colitogenic pathobionts and pathogenic T cells.

Virus-like particles (VLPs) are nanoscale biological structures consisting of viral proteins assembled in a morphology that mimic the native virion but do not contain the viral genetic material. The possibility of chemically and genetically modifying the proteins contained within VLPs makes them an attractive system for numerous applications. As viruses are potent immune activators as well as natural delivery vehicles of genetic materials to their host cells, VLPs are especially well suited for antigen and drug delivery applications. Despite the great potential, very few VLP designs have made it through clinical trials. In this review, we will discuss the challenges of developing VLPs for antigen and drug delivery, strategies being explored to address these challenges, and the genetic and chemical approaches available for VLP engineering.

Protein therapeutics is a rapidly growing segment of the pharmaceutical market. Currently, the majority of protein therapeutics are manufactured in mammalian cells for their ability to generate safe and efficacious human-like glycoproteins. The high cost of using mammalian cells for manufacturing has motivated a constant search for alternative host platforms. Insect cells have begun to emerge as a promising candidate, largely due to the development of the baculovirus expression vector system. While there are continuing efforts to improve insect-baculovirus expression for producing protein therapeutics, key limitations including cell lysis and the lack of homogeneous humanized glycosylation still remain. The field has started to see a movement toward virus-less gene expression approaches, notably the use of clustered regularly interspaced short palindromic repeats to address these shortcomings. This review highlights recent technological advances that are realizing the transformative potential of insect cells for the manufacturing and development of protein therapeutics.

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The subgingival microbiome is one of the most stable microbial ecosystems in the human body. Alterations in the subgingival microbiome have been associated with periodontal disease, but their variations over time and between different subgingival sites in periodontally healthy individuals have not been well described. We performed extensive, longitudinal sampling of the subgingival microbiome from five periodontally healthy individuals to define baseline spatial and temporal variations. A total of 251 subgingival samples from 5 subjects were collected over 6–12 months and deep sequenced. The overall microbial diversity and composition differed significantly between individuals. Within each individual, we observed considerable differences in microbiome composition between different subgingival sites. However, for a given site, the microbiome was remarkably stable over time, and this stability was associated with increased microbial diversity but was inversely correlated with the enrichment of putative periodontal pathogens. In contrast to microbiome composition, the predicted functional metagenome was similar across space and time, suggesting that periodontal health is associated with shared gene functions encoded by different microbiome consortia that are individualized. To our knowledge, this is one of the most detailed longitudinal analysis of the healthy subgingival microbiome to date that examined the longitudinal variability of different subgingival sites within individuals. These results suggest that a single measurement of the healthy subgingival microbiome at a given site can provide long term information of the microbial composition and functional potential, but sampling of each site is necessary to define the composition and community structure at individual subgingival sites.

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Strains and plasmids used in this study are listed in Table S1. Primers were synthesized by Integrated DNA Technologies (Coralville, IA) and listed in Table S2. All plasmids were constructed using either the DNA assembler method based on homologous recombination in W303-1a 1 or ligating the target genes into digested plasmid backbones in Escherichia coli strain Mach1 (Thermo Fisher Scientific, Waltham, MA). Restriction enzymes and DNA polymerase were purchased from New England Biolabs (Beverly, MA). Yeast-assembled plasmids were isolated using Zymoprep II Yeast Plasmid Miniprep kit (Zymo Research, CA). Plasmids were isolated from E. coli using QIAprep Spin Miniprep Kits (Qiagen, Hilden, Germany). E. coli was grown in LB medium (Sigma-Aldrich, St. Louis, MO) at 37°C in an orbital shaker at 225 RPM. LB was supplemented with 50 µg/mL ampicillin for plasmid propagation. Plasmid Construction Construction of p426-XR-XDH-XK: Plasmid p426-XR-XDH-XK contains expression cassettes for XR, XDH, XK, and the KanMX resistance gene flanked by δ homology sequences that enable genomic integration into yeast δ sites. Fragments Delta-Left, Delta-Right, promoters (TEF1, PGK1 and PYK1), and terminators (ADH1, CYC1 and ADH2) were PCR amplified from S. cerevisiae W303-1a genomic DNA (Table S2). The KanMX cassette was amplified using plasmid pRS-TEFp-KanMX-TEFt as a template (a gift from Zengyi Shao, Iowa State University). The genes for XR, XDH, and XK were amplified from S. stipitis genomic DNA. All PCR fragments were assembled into plasmid pRS426 linearized with XhoI and NotI using the DNA assembler method. W303-1a cells were transformed with p426-XR-XDH-XK by electroporation and plated on SC-URA. Construction of single gene plasmids p424-XR, p425-XK, and p426-XDH: Expression cassettes for XR, XK, and XDH were PCR-amplified from plasmid p426-XR-XDH-XK and cloned into plasmids pRS424, pRS425, and pRS426, respectively, by digestion and ligation using restriction enzymes listed in Table S2. W303-1a cells were transformed with p424-XR, p425-XK, and p426-XDH by electroporation and plated on SC-TRP, SC-LEU, and SC-URA, respectively. Construction of p426gRNA-Delta and p423gRNA-Delta plasmids: The p426gRNA-Delta and p423gRNA-Delta plasmids generate a guide RNA (gRNA) for the Cas9 nuclease that is specific for the S. cerevisiae δ site with the sequence tatactagaagttctcctcg. To produce plasmid p426gRNA-Delta, fragments gRNA F1 and gRNA F2 (Table S2) were PCR amplified from SNR52p-gRNA.CAN1.Y-SUP4t 2 (a gift from George M. Church, Addgene plasmid #43803), assembled by overlap extension PCR 3 , and ligated into p426-SNR52p-gRNA.CAN1.Y-SUP4t after digestion with NheI and BsrGI. Plasmid p423gRNA-Delta was generated by PCR amplification of the gRNA fragment using g426RNA-Delta plasmid as template (Table S2). The amplified fragment, gRNA F3, was ligated into p423 after digestion with SalI and NotI. Construction of p426-d-XDH-d: Fragments Delta-Left and Delta-Right were PCR amplified from S. cerevisiae W303-1a genomic DNA (Table S2). The XDH expressio...